Vaccinia virus and more recently other poxviruses have been used for the insertion and expression of foreign genes. The basic technique of inserting foreign genes into live infectious poxvirus involves recombination between pox DNA sequences flanking a foreign genetic element in a donor plasmid and homologous sequences present in the rescuing poxvirus (Piccini et al., 1987).
Specifically, the recombinant poxviruses are constructed in two steps known in the art and analogous to the methods for creating synthetic recombinants of the vaccinia virus described in U.S. Pat. No. 4,603,112, the disclosure of which patent is incorporated herein by reference.
First, the DNA gene sequence to be inserted into the virus, particularly an open reading frame from a non-pox source, is placed into an E. coli plasmid construct into which DNA homologous to a section of DNA of the poxvirus has been inserted. Separately, the DNA gene sequence to be inserted is ligated to a promoter. The promoter-gene linkage is positioned in the plasmid construct so that the promoter-gene linkage is flanked on both ends by DNA homologous to a DNA sequence flanking a region of pox DNA containing a nonessential locus. The resulting plasmid construct is then amplified by growth within E. coli bacteria (Clewell, 1972) and isolated (Clewell et al., 1969; Sambrook et al., 1989).
Second, the isolated plasmid containing the DNA gene sequence to be inserted is transfected into a cell culture, e.g. chick embryo fibroblasts, along with the poxvirus. Recombination between homologous pox DNA in the plasmid and the viral genome respectively gives a poxvirus modified by the presence, in a nonessential region of its genome, of foreign DNA sequences. The term "foreign" DNA designates exogenous DNA, particularly DNA from a non-pox source, that codes for gene products not ordinarily produced by the genome into which the exogenous DNA is placed.
Genetic recombination is in general the exchange of homologous sections of DNA between two strands of DNA. In certain viruses RNA may replace DNA. Homologous sections of nucleic acid are sections of nucleic acid (DNA or RNA) which have the same sequence of nucleotide bases.
Genetic recombination may take place naturally during the replication or manufacture of new viral genomes within the infected host cell. Thus, genetic recombination between viral genes may occur during the viral replication cycle that takes place in a host cell which is co-infected with two or more different viruses or other genetic constructs. A section of DNA from a first genome is used interchangeably in constructing the section of the genome of a second co-infecting virus in which the DNA is homologous with that of the first viral genome.
However, recombination can also take place between sections of DNA in different genomes that are not perfectly homologous. If one such section is from a first genome homologous with a section of another genome except for the presence within the first section of, for example, a genetic marker or a gene coding for an antigenic determinant inserted into a portion of the homologous DNA, recombination can still take place and the products of that recombination are then detectable by the presence of that genetic marker or gene in the recombinant viral genome.
Successful expression of the inserted DNA genetic sequence by the modified infectious virus requires two conditions. First, the insertion must be into a nonessential region of the virus in order that the modified virus remain viable. The second condition for expression of inserted DNA is the presence of a promoter in the proper relationship to the inserted DNA. The promoter must be placed so that it is located upstream from the DNA sequence to be expressed.
The technology of generating vaccinia virus recombinants has recently been extended to other members of the poxvirus family which have a more restricted host range. The avipoxvirus, fowlpox, has been engineered as a recombinant virus expressing the rabies G gene (Taylor et al., 1988a,b). This recombinant virus is also described in PCT Publication No. WO089/03429. On inoculation of the recombinant into a number of non-avian species an immune response to rabies is elicited which in mice, cats and dogs is protective against a lethal rabies challenge.
Malaria today still remains one of the world's major health problems. It is estimated that 200-300 million malaria cases occur annually while 1-2 million people, mostly children, die of malaria each year. Malaria in humans is caused by one of four species of the genus Plasmodium--P. falciparum, P. vivax, P. malariae, and P. ovale. Clinically, P. falciparum is the most important human Plasmodium parasite because this species is responsible for most malaria fatalities.
A Plasmodium falciparum infection starts with the bite of an infected female Anophele mosquito. Its saliva contains sporozoites that migrate in the blood vessels to reach their first targets, the hepatocytes. After invasion, the sporozoites undergo a first multiplication stage lasting between five to seven days (exoerythrocytic phase or liver stage). Each hepatocyte can release 10,000 to 40,000 merozoites into the blood stream. Merozoites infect the second cellular target, the erythrocytes, where they multiply during a 48 to 72 hour cycle (erythrocytic stage). Each infected erythrocyte can release 16 merozoites able to infect new erythrocytes. The clinical symptoms of malaria appear during the blood stage infection. Infected erythrocytes can also produce gametocytes that mature and fuse in the mosquito midgut to form the zygotes. The zygotes evolve into ookinetes that develop into oocystes which, after infection of epithelial cells, produce sporozoites. The sporozoites migrate into the salivary glands from where they can initiate a new human infection.
The acquisition of protective immunity against malaria in naturally infected people is a slow process requiring multiple infections and is Plasmodium falciparum specific. The components that elicit immunity and the exact nature of this protective immune response are largely unknown but include activation of both specific and non-specific humoral and cellular mechanisms directed against a variety of sporozoite, liver stage and erythrocytic stage antigens.
MSA1 (Merozoite Surface Antigen 1), also referred to as PMMSA, p195 and PSA, is the best characterized biochemically and immunologically asexual erythrocytic antigen. It has been used alone and in combination with other blood stage antigens to vaccinate humans and monkeys against malaria.
MSA1 is a schizont surface glycoprotein which is proteolytically cleaved at the time of schizont rupture to generate the majority of the antigens detected on the extracellular surface of the merozoites (Lyon et al., 1987; Holder, 1988a). During merozoite invasion in vitro all but the C-terminal 19 kd of MSA1 are shed. The precise role of MSA1 is still unknown. Polymorphism has been reported in this protein among various Plasmodium falciparum isolates and constant, semi-constant and variable regions have been localized within the molecule. A more precise analysis determined that the polymorphism could be reduced to a dimorphism (Tanabe et al., 1987) even if three distinct versions of one of the variable regions have been identified (Peterson et al., 1988).
MSA1 is probably one of the strongest malarial vaccine candidates. This is supported by ten different reports of vaccine trials in which primates have been immunized with complete MSA1 or derived peptides and challenged with infected erythrocytes (Perrin et al., 1984; Hall et al., 1984; Cheung et al., 1986; Siddiqui et al., 1986; Siddiqui et al., 1987; Patarroyo et al., 1987; Patarroyo et al., 1988; Holder et al., 1988; Knapp et al., 1988; Herrera et al., 1990).
In the search for a malaria vaccine, the possibility of using a live recombinant vaccine has not been extensively studied. Indeed, the majority of the malaria vaccines are purified native antigens or synthetic peptides derived from them.
It can be appreciated that provision of a malaria recombinant poxvirus, and of vaccines which provide protective immunity against Plasmodium infections, would be a highly desirable advance over the current state of technology.